The long-term objective of this proposal is to better understand the mechanism of hematopoietic stem cell self-renewal and its implication in hematological malignancies. To achieve this, I plan to focus on the function of the human Piwi stem cell proteins and their piRNA partners during normal and malignant hematopoiesis. My hypothesis is that the human Piwi homologs, HIWI and HILI, and their associated piRNAs are necessary for the multipotentency and long-term maintenance of hematopoietic stem cells (HSCs). The misregulation of human PIWI proteins during hematopoiesis can lead to leukemia. To test this hypothesis, I propose the following aims: (Aim1) Identify HIWI- and HILI-associated piRNAs in normal hematopoietic precursors and primary human leukemia cells. This aim will correlate HIWI and HILI function with piRNA partners during hematopoiesis and will also potentially identify piRNAs that are unique to normal hematopoietic precursors and to leukemic cells. (Aim2) Characterize the expression of HIWI, HILI, and their associated piRNAs with respect to hematopoietic lineage restriction. I will screen CD34+ cells and lineage-restricted hematopoietic progenitor cells for the expression of HIWI, HILI, and their associated piRNAs. These studies will allow us to determine the relationship of HIWI, HILI, and their piRNA partners during normal differentiation down the various hematopoietic lineages. (Aim3) Examine HIWI, HILI, and piRNA function by characterizing the effect of overexpression and knockdown of HIWI and HILI on HSC division. I will examine the effect of overexpressing HIWI and HILI on the proliferation ability and possible reversion to a leukemia-like state of lineage-restricted hematopoietic progenitor cells. I will also determine the impact on stem-cell phenotypes by knocking down HIWI and HILI in CD34+ cells and leukemia cells. In addition, this aim will allow me to examine the dose effect of HIWI and HILI on piRNA expression in normal and malignant hematopoiesis. Disease Relevance: Our work will give critical insight into how PIWI proteins and their partner molecules, piRNAs, contribute to the onset and maintenance of leukemia. We believe that understanding the role of Piwi/piRNA complexes in blood stem cells is a prerequisite for the effective treatment of leukemia.